PROJECT SUMMARY/ ABSTRACT Early-life exposure to Superfund metal toxicants such as lead (Pb), arsenic (As), cadmium (Cd) and Manganese (Mn) has been associated with worse cognitive function during aging and is suspected of contributing to the development of neurodegenerative diseases, such as Alzheimer?s disease. However, the biological mechanisms underlying the associations remain poorly understood. Mammalian cells, including neurons and neural stem cells, secrete into the extracellular milieu a variety of tiny membrane-encapsulated vesicles. These extracellular vesicles (EVs) contain functional signaling molecules that can be taken up by recipient cells to mediate intercellular communication. One such group of signaling molecules is microRNAs, which function as master tuners of gene expression by degrading target mRNA and/or inhibiting translation of the message. Since EVs are encapsulated by a lipid bilayer membrane, molecules such as microRNAs enclosed in the vesicles are protected from nuclease-mediated degradation and thus are thus very stable. As a result, EV microRNAs can be easily detected and quantitated in biological fluids such as plasma/serum and have been used as novel biomarkers for a variety of human diseases. Although some limited studies have explored the role of EV microRNAs in neural cells, no studies have examined the role of EV microRNAs on cognitive function in the context of environmental exposures such as metal toxicants. We hypothesize that metal exposures in early life alter EV microRNAs in the brain and that these changes in EV microRNAs affect the function of neurons and neural stem cells to accelerate cognitive aging. We propose three interconnected Specific Aims to test this hypothesis. Aim 1 will determine the effects of exposures to individual metal exposures (Pb, As, Mn, and Cd) as well as ?real-world? metal mixtures (pre- and post-remediation water samples collected at the San Luis Valley Superfund site) on developing human fetal brain organoids. Aim 2 will determine the effects of early-life exposure to individual metals (Pb and As) as well as to the real-world metal mixtures on EV miRs and the cognitive function of mice later in life. Aim 3 will determine the functional role of selected EV microRNAs in modulating functions of brain organoids and cognitive function in mice. Our highly multidisciplinary study integrating mouse models, human epidemiology, and functional cellular studies seeks to establish EV microRNAs not only as novel biomarkers for metal exposure-related cognitive function, but also as a mechanistic basis for metal- induced neurotoxicity and cognitive impairment. This project links with the MEMCARE-SRC by complementing human studies in Project 1 and seeking to identify biologic mechanisms for health effects of water contamination at Superfund sites.